GENE EXPRESSION

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18.3: GENE EXPRESSION

Title: Manipulation of DNA

Introduction:

I have been assigned to look into the production of GM foods and to see whether techniques that have been developed works on the wanted organism. I am to insert genes for water tolerance into a bacterium so it will infect and hopefully protect a plant. To see whether this works the marker gene is for the production of a protein that glows in the dark

Aim: I will be changing the genes of a bacterium to see whether it will glow in the dark.

BACKGROUND INFORMATION

With this practical, the aim is to add a gene to bacteria which codes for Green Fluorescent Protein (GFP). This gene is found naturally in a jellyfish (Aequorea victoria) which causes the jellyfish to fluoresce and glow in the dark.

pGLO plasmid – contains the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also has a gene regulation system that can be used to control expression of the fluorescent protein. The gene can be switched on by adding the sugar ARABINOSE to the cell’s nutrient medium.

The selection of cells that have been transformed with pGLO DNA is done by growth on antibiotic plates. The transformed cells will appear white (wild type phenotype) on plates not containing arabinose and fluorescent green when arabinose is present.

The pGLO plasmid contains the gene for GFP but also the gene for β-lactamase, which provides resistance to ampicillin. Β-lactamase inactivates the ampicillin present on the nutrient agar, allowing bacterial growth. Only transformed bacteria will grow on plates that contain ampicillin. Only a very small percentage of cells take up the plasmid DNA and are transformed.

Transformation solution – contains calcium chloride. The calcium cation neutralizes the repulsive negative charges of the phosphate backbone of the DNA and phospholipids of the cell membrane, allowing DNA to enter the cells.

Heat shock – increases permeability of the cell membrane to DNA. The mechanism is not known but the duration for heat shock is critical and has been opitmized for the type of bacteria used and the transformation conditions employed.

Recovery – this period follows the addition of LB nutrient broth allows the cells to grow and express the ampicillin resistance protein β – lactamase, so that the transformed cells survive on the ampicillin selection plates.

Media – known as LB (named after Luria and Bertani).Made from an extract of yeast and an enzymatic digest of meat byproducts, which provides carbohydrates, amino acids, nucleotides, salts and vitamins for bacterial growth.

Method